CN1918309A - Bacterial oxidation of sulphide ores and concentrates - Google Patents
Bacterial oxidation of sulphide ores and concentrates Download PDFInfo
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- CN1918309A CN1918309A CNA2004800417211A CN200480041721A CN1918309A CN 1918309 A CN1918309 A CN 1918309A CN A2004800417211 A CNA2004800417211 A CN A2004800417211A CN 200480041721 A CN200480041721 A CN 200480041721A CN 1918309 A CN1918309 A CN 1918309A
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- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B15/00—Obtaining copper
- C22B15/0063—Hydrometallurgy
- C22B15/0065—Leaching or slurrying
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B15/00—Obtaining copper
- C22B15/0063—Hydrometallurgy
- C22B15/0065—Leaching or slurrying
- C22B15/0067—Leaching or slurrying with acids or salts thereof
- C22B15/0071—Leaching or slurrying with acids or salts thereof containing sulfur
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- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
- C22B3/18—Extraction of metal compounds from ores or concentrates by wet processes with the aid of microorganisms or enzymes, e.g. bacteria or algae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/20—Recycling
Abstract
A bacterial culture for use in the bacterial oxidation of sulphide ores and concentrates, the bacterial culture identified by AGAL deposit Accession No. NM99/07541 or having been adapted therefrom, the bacterial culture further containing one or more strains of both Sulfobacillus and Thermoplasma. The present invention further provides a process for the bacterial oxidation of sulphide ores and concentrates characterised in that the ore or concentrate is leached using such a bacterial culture.
Description
Invention field
The present invention relates to use the improved sulfide ore (sulphide ores) of bacterial cultures and the bacterial oxidation of refined ore (concentrates).
Bio-oxidation process of the present invention specifically is applied to contain the ore of chalcopyrite and the bacterial oxidation of refined ore.
Background technology
Bacterial oxidation is successfully used to the processing of arsenopyrite (arsenopyrite), pyrite (pyrite), pyrrhotite (pyrrhotite), covellite (covellite) and copper glance (chalcocite) ore and refined ore for many years, but this processing does not comprise chalcopyrite (CuFeS
2) oxidation of ore and refined ore.
Bacterial mixture of the prior art is used to help the sulfide ore except chalcopyrite ore and refined ore and the oxidation of refined ore, and these bacterial mixtures use various bacteria combination.For example, the mixt bacteria culture of South Africa Gencor Limited use mainly comprises thiobacillus ferrooxidant (Thiobacillus ferrooxidans), thiobacillus thiooxidans (Thiobacillus thiooxidans) and ferricoxidans (Leptospirillum ferrooxidans).The Gencor culture is made up of the mixed bacterial of mesophilic bacterium, these bacteriums work in 35 ℃ to 45 ℃ temperature range (Dew and Miller, 1997).
And the Finnish patent application 953488 of Gencor Limited discloses uses thiobacillus ferrooxidant, thiobacillus thiooxidans and ferricoxidans preferred pH 3 times the ore that preferably is crushed to below the 6mm to be realized oxidation.
The employed bacterial cultures of BacTech (Australia) Pty Ltd referring to for example United States Patent (USP) 5429659, is a kind of bacterial cultures of moderate thermophile, and it is worked in 46 ℃ to 50 ℃ temperature range.This culture is described (Brierley and Brans 1994) by called afters such as Barrett " M4 " (1998) and by (1988) such as Nobar.
The MINBAC technology utilization of being developed by the Mintek-Anglo American Corporation that is positioned at South Africa Randburg a kind of mesophilic bacterium mixed culture, this culture comprises thiobacillus ferrooxidant/ferricoxidans (Brierley and Brans 1994).
The present bacterial cultures that uses, if not with ore or refined ore superfine grinding (P80＜20 μ m) to help bacterial oxidation, perhaps use very long extraction time (leach times) to realize oxidation, just can not produce commercial acceptable effect.The extraction time that surpasses 100 days is unrare.
Present trend tends to use higher temperature to promote the iron oxidation.But used high temperature makes needs cooling later in oxidation, and for example reactor of the stainless steel making of surgical grade of certain material need be provided.These two kinds of situations all can increase the cost of operation.
Be used for from the secondary copper product that is easier to oxidation for example covellite and copper glance reclaim the bacterium technology of copper, (heap leach) is the most commonly used in dump leaching.This technology comprises that the ore storage that will pulverize is placed on the bed course (pad) of special non-property, and the design of this bed course makes and is gathered in a bit from the mother liquor of piling under the row, from then on enters in the collecting tank.Come to reclaim metal by precipitation, solvent extraction and/or electrowinning from mother liquor (pregnant liquor) lining.
Go out in order successfully to carry out dump leaching, it is crucial keeping the complete of heap.The principal element of the stability of decision heap is the grinding particle size (crush size) of ore.The pulverizing of ore must reach such degree, be that ore is enough thin, make leaching agent to permeate well to pass heap and too much channel (channeling) do not take place, and keep void space simultaneously that it is crucial that void space is discharged for good air diffuser and leaching agent.If ore is pulverized meticulously, passing the heap infiltration will be very slow.Void space will be not enough, and can pile the draining deficiency, causes heap to go up and form head (phreatic head) under pond (pooling) and the highland.On the other hand, if the granularity of ore is too thick, the speed of then piling draining is fast, and the level that GOLD FROM PLATING SOLUTION belongs to is low, and because ore is destroyed through the biological and chemical process, pile structure can damage.Under many circumstances, with tackiness agent, sulfuric acid and water the ore of pulverizing is gathered group before piling up, make that granularity and the acid in the heap is more evenly distributed.
Before accumulation, usually layer of displacement is placed on the bed course, layer of displacement generally by non-reacted rock for example quartzite constitute, guarantee that mother liquor discharges fully.With acidifying bacterium liquid pouring heap, acidifying bacterium liquid serves as leaching agent copper is leached from ore.The bacterium of using in the dump leaching generally is aerobic, therefore needs oxygen.Oxygen can be pressed in the heap by low pressure blower, and perhaps, because the chimneyeffect (chimney effect) that is taken place when bacterial oxidation ore and heat production, air can be inhaled in the heap.
Geocoat method (referring to WO 96/38381) is a kind of version of dump leaching, and by the marketization of U.S. Geobiotics company.This method comprises from sulfide ore generation refined ore, it is covered on rock pulverizing, that size is certain form the heap that can carry out bacterial oxidation.Importantly, the invention of this method it seems be one be intended to guarantee to pile in the effort of sufficient air and fluid flow and heat radiation.
Hillock leaching (damp leach) and dump leaching are closely similar, generally only are used for the lower ore of grade.Hillock leaches and often to be counted as the adjoint process of dump leaching, and itself is not sufficient to as an engineering independently.Crucial is, when having to exploitation and pile up ettle or low grade ore, and when almost not doing ground in advance and preparing, can extract some object of values (value) in these materials.Have native bacterium (indigenous bacteria) in the heap, what need only is the activity that improves them.This can realize by adding acid and nutrition in pouring solution, just as dump leaching.Difference is cost.
Before piling up, seldom or not carry out fragmentation.Only need carry out minimum bed course preparation, and not have artificial aeration.
On cost, complexity and efficient, the bucket formula leaches the intermediate form that (vat leach) can regard dump leaching and slot type leaching (tank leach) as.In bucket formula leaching process, material to be processed is immersed in the infusion solution fully, is not stirred, and is not stirred significantly at least, although because some stirrings may take place in flowing of air and/or solution.This method is better than dump leaching or hillock and leaches part and be to realize the complete wetting of mineral and avoid channel.Thinner grinding particle size also can be handled in bucket better, though since the perviousness of air and solution need fineness limited.When surpassing this limit, the material that just needs a person with the qualifications of a general is suspended in the solution.Use if bucket is only made single, can build up them the dam (lined dams) of lining cutting, to an angle lapping tiltedly to allow the circulation and the recovery of leach liquor.Make nonexpondable bucket and then need firmer structure, for example concrete or brick.Ventilation can be managed by latent (submerged) and be realized, perhaps also can be by intermittently bucket being discharged water, and the leach liquor that air is retreated is drawn onto in the ore.
Slot type leaches, and as its name suggests, is included in that the ore pulp to ventilation carries out the bacterium leaching in the groove of stirring.Imagining this technology may be very similar with the biology leaching of base metal, but the business-like system that is used for copper does not up to now also set up.
The data presentation that can get can prediction can make fund and running cost too high to the related expense of refined ore super fine powder (ultra-fine milling) (P80＜30 μ m).
One of purpose of the present invention is to overcome the relevant the problems referred to above of prior art, perhaps is at least prior art a useful replacement scheme is provided.
The discussion of front background technology only is in order to help to understand the present invention.Be to be understood that above-mentioned discussion and do not mean that approval or admit that any material that is mentioned has constituted the part of general common knowledge before the priority date of the present invention.
In whole specification sheets, unless context has requirement in addition, word " formation " (comprise), version for example " formation " (comprises) or " formation " (comprising), should be understood to comprise described integral body or whole group, but do not get rid of other any integral body or whole group.
In whole specification sheets, when mentioning certain bacterium, should be understood to also comprise its subspecies.
In whole specification sheets, ore is understood that such material: it is from surface separation, and does not accept any processing to improve metal concentration.Refined ore is to produce like this: by making ore experience treating processes, be generally gravity or flotation, to improve the volume of required concentration of metal and reduction material.Next through handling this material to reclaim required metal.
Of the present invention open
According to the present invention, the bacterial cultures of the bacterial oxidation that is used for sulfide ore and refined ore is provided, this bacterial cultures be identified by AGAL preservation registration number NM99/07541 or by adaptation and come, this bacterial cultures also contains one or more bacterial strains of sulfuration Bacillaceae (Sulfobacillus) and Thermoplasma (Thermoplasma) simultaneously.
According to the present invention, the bio-oxidation process of a kind of sulfide ore and refined ore also is provided, it is characterized in that using the bacterial cultures that identified by AGAL preservation registration number NM99/07541 or by and adapt to and the bacterial cultures that comes leaches this ore or refined ore, this bacterial cultures also contains one or more bacterial strains of sulfuration Bacillaceae and Thermoplasma simultaneously.
In a kind of form of the present invention, described sulfide ore or refined ore contain chalcopyrite (chalcopyrite).
Leaching (leach) used in the method for the present invention can be carried out to be selected from by the form of the following group of forming:
Dump leaching (heap leach),
Slot type leaches (tank leach),
Bucket formula (vat) leach and
Hillock (dump) leaches.
Preferably, described bacterial cultures is non-indigenous (indigenous) to ore or the refined ore of wanting oxidation.
When the ore that is provided or refined ore have the P of being equal to or greater than
80When the grinding of 90 μ m or grinding particle size, the oxidation in sulfide ore and refined ore of bacterial cultures of the present invention and method is effective.Preferably, the ore or the refined ore that are provided have the P of being equal to or greater than
80The grinding of 75 μ m or grinding particle size.
In a form of the present invention, this culture can be worked in the oxidation of sulfide ore and refined ore in 45 ℃ to 90 ℃ temperature range.Preferably, this culture can be worked in the oxidation of sulfide ore and refined ore in 45 ℃ to 65 ℃ temperature range.
Brief description
Describe the present invention with reference to institute's accompanying drawing below, only make usefulness for example with reference to accompanying drawing.Wherein:
Fig. 1 is the diagram by the denaturing gradient gel of the sample of 6 bacterial culturess of the present invention of three kinds of different methods processing.
Invention is described
In order to cultivate the cultivation thing that to process chalcopyrite ores and refined ore, sought a kind of native bacterium of brass mineral and cultivated thing. Thing is cultivated in the separation that original inhabitants' bacterial cultures is better than modifying usually, because original inhabitants' cultivation thing has adapted to relevant toxin and the mineral component of particular ore, thereby produces more effective, the better bacterium bacterial strain of restorability.
Cultivating the native bacterium of chalcopyrite ores cultivates thing and detects its autochthonal ore/refined ore of its oxidation and the ability of other chalcopyrite ores and refined ore. In this working procedure, from produce the chalcopyrite (CuFeS of gained the base metal ore from Canadian New Brunswick2) cultivated a bacterium stock culture in the refined ore. Separate after this bacterial cultures, its autochthonal ore and refined ore and multiple other ores and refined ore are tested. When the cultivation thing of the different mineral of test, any autochthonal bacterium if it can be worked under experimental condition, can be worked competitively with the cultivation thing of introducing, and under this environment, not only can survive but also vigorous the growth, namely be added in original stock culture. Like this, As time goes on, any autochthonal bacterium in tested ore or the refined ore all is incorporated in this cultivation thing. In addition, this stock culture under the different temperatures in 40 to 90 ℃ of scopes with pH 0.8-2.2 scope in different acidity under carried out successful cultivation. The applicant believes that 0.5 to 3.0 pH boundary is not irrational. Stock culture is successfully tried out and is stirred in the stirred-tank reactor and help post leaching in ventilating column in ventilation. Under various temperature and in multiple ore and the refined ore, successfully tried out stock culture.
Bacterial cultures of the present invention is comprised of multiple iron, sulfide and sulfur-oxidizing bacteria, works in the pH scope of these bacteriums between can the temperature and 0.5 to 3.0 between 35 ℃ to 90 ℃, although optimum pH is between 0.8 to 2.5. The bacterial cultures that mixes has been considered to comprise simultaneously that sulfuration Bacillus and pyrogen body belong to one or more representatives of (Thermoplasma), the bacterium that also may have simultaneously other kinds. These bacteriums may include but not limited to, the ferrous Thiobacillus of sulfobacillus thermosulfidooxidans (Sulfobacillus thermosulfidooxidans), Thiobacillus caldus and/or oxidation.
Mixed cell of the present invention is cultivated thing and is deposited in Government Of Australia assay laboratory (Australian Government Analytical Laboratories) according to the budapest treaty needs, and preserving number is NM99/07541.
In the bacterium oxidation process of ore of the present invention and refined ore, above-mentioned storage with bacterial cultures generally speaking and the ore that will be leached or the sample combination of refined ore, as an adaptation process. It is non-indigenous to this ore or refined ore that this storage is cultivated thing with (stock). Then the cultivation thing that adapts to can be inoculated on its ore that adapts to or the concentrate clitter. As selection, also can use other Leaching way as the cultivation thing and the means that ore or refined ore contact that make adaptation, comprise bucket, groove or hillock.
To describe the present invention by some informative embodiment below, above-described generality of the present invention is also unrestricted.
Embodiment 1
Before any material was tested, at first making as described above, stock culture was adapted to target material.Means are the improved 0K substratum of 2700ml (1.0g/L ammonium sulfate, 0.5g/L ortho-phosphoric acid dipotassium, 0.16g/L magnesium sulfate heptahydrate, pH 1.6-1.8) to be added be preheating to temperature required stirring aeration-agitation groove type reactor.(the P that has added pulverizing in the improved 0K substratum
80＜45 μ m) sample of the 150g of test materials if desired, is turned down pH between 1.6 and 1.8 with the vitriol oil.In this slurry, add 300ml storage inoculum slurry samples.Stir the speed ventilation of reactor with the 1L/min/L slurry.According to design, storage is non-indigenous with inoculum for the ore of test materials.Continue adaptive process, reach 100% or reach steady up to the level of (the reporting to solution) of solution stripping associated metal.Use the metal level in inductively coupled plasma (ICP) the analytical solution sample, wherein the pH with vitriol oil adjusting ore pulp makes that its pH is between 1.6 to 1.8.Except the metal level of solution stripping, further monitor adaptation/process of the test according to redox potential (ORP), concentration of iron and oxyty (DO).
In case culture is adapted to the target mineral, it just is used as the inoculum that further stirs the test of aeration-agitation groove type reactor, or the inoculum of conduct heap or column test.The acid basic nutritive medium that contains ammonium sulfate, potassium orthophosphate and sal epsom by adding further dilutes the bacterial inoculum that adapts to.Above-mentioned nutraceutical concentration is between laboratory test and commercial operation in the solution, and has variation between the operation of different commerce.In all cases, oxidising process all monitor by level, pH, ORP, concentration of iron and the DO content of the metal by the solution stripping.
The tested a plurality of chalcopyrite samples that contain that are used for from different location, the whole world of bacterial cultures of the present invention.Below table 1 the chalcopyrite refined ore that uses bacterial cultures of the present invention and the mineralogy and the source of ore have been described.
Table 1
| Sample | Mineralogy | The source |
| A | Chalcopyrite copper ore concentrates stone. | The U.S. |
| B | The concentrated molybdenum ore stone that contains low-level copper in the chalcopyrite. | Canada |
| C | The refined ore that mainly comprises chalcopyrite (35%) and cubanite (cubanite) (17%) and more a spot of pyrrhotite (pyrrhotite) (10%) and a spot of pentlandite (3%) and zink sulphide (sphalerite) (3%). | Canada |
| D | Niccolite (Copper Nickel concentrate) refined ore, wherein copper exists with chalcopyrite (18.5-28.5%) and cubanite (15.8-30.8%).Nickel exists with pentlandite (17.7-10.4%), is replaced by violarite (violarite) sometimes. | The U.S. |
| E | Three kinds of copper ore concentrates stones comprise chalcopyrite, pyrite and a small amount of purple copper. | Canada |
| F | The copper ore concentrates stone of forming by copper glance (14%), chalcopyrite (10%), purple copper (1%) and pyrite (1%) | South Africa |
| G | Sample i and ii are ore sample, and sample iii is the refined ore sample.Sulfide mineral mainly is pentlandite (pentlandite), chalcopyrite and pyrrhotite. | The West Australia |
The ordinary test step
All operations to mineral samplers all carries out in stirring the ventilation groove type reactor.The solid density of each test is 10%w/v, by speed bubbling (sparging) ventilation of the slurry in every liter of reactor of 1L air per minute.Replenished the vaporization losses that heats and ventilate and cause because of to slurry before test sheet, this realizes by adding tap water.All slurries all are to prepare in a kind of initial pH is 1.0 proprietary nutritional medium.Sampling comprises iron, copper and other associated metal ions in the analytical solution.In addition, all monitored and record of redox potential (ORP), pH, iron ion and dissolved oxygen level.Utilize copper to discharge the carrying out of monitoring test, in case its reach plateau or approximately reach the solution stripping copper 100%, think that then test finishes.In case finish, with ore pulp press filtration (pressure filtered), analyze final leach liquor, filter cake (filter cake) acidifying water washing after drying.Dried filter cake is weighed and analyze remnants, to carry out metal balance.
Table 2 summary has also shown raw ore analysis, the result of sreen analysis and the result after the oxidation.
Table 2
| Sample | Raw ore (Head) is analyzed | T℃ | Leach fate | Leach the back result | |||
| Sreen analysis | Fe％ | Cu％ | S Always％ | The %Cu that leaches | |||
| A | P 81＜90μm | 28.60 | 29.40 | 32.1 | 48 | 36 | 96.6 |
| B | P 85＜90μm | 2.85 | 1.95 | 37.6 | 48 | 20 | 96.9 |
| C | P 80＜75μm | 27.30 | 20.97 | 27.37 | 48 | 22 | 98.0 |
| D | P 80＜75μm | 26.30 | 12.80 | 25.1 | 48 | 27 | 95.0 |
| Ei | P 84＜75μm | 15.00 | 2.87 | 13.9 | 48 | 28 | 99.3 |
| Eiii | P 78＜75μm | 26.6 | 4.62 | 34.4 | 48 | 28 | 99.3 |
| F | P 80＜43μm | 6.79 | 28.5 | 10.2 | 60 | 14 | 95.3 |
| Gi | P 80＜75μm | 17.8 | 1.18 | 7.88 | 48 | 14 | 98.8 |
| Gii | P 80＜75μm * | 45.1 | 6.82 | 34.8 | 50 | 10 | 98.0 |
| Giii | P 80＜75μm | 18.2 | 0.1 | 3.11 | 50 | 8 | 97.3 |
| H | P 80＜75μm * | 23.8 | 19.7 | 36.7 | 48 | 15 | 99.2 |
*" receive " nominal particle size of (as received) refined ore.
A plurality of samples of the bacterial cultures of adaptation of the present invention are cultivated in general 30 ℃ to 65 ℃ temperature range, work although the inventor mentions under up to about 90 ℃ temperature.Separated and be ready for 16S rRNA order-checking and identify from the sample of each culture.Sample before the RNA order-checking is prepared to be to use 3 kinds of different methods to carry out.Method of using and 16S rRNA order-checking gained result are as follows.
Method
6 samples (called after SN45, SM45, PO45, SS 45, RH 14K and 014A respectively) have been tested.
Go up with top speed sample mix 30 minutes at hand vibrator (hand shaker), do following processing then:
A. vibration. get the sample 500 μ l that vibrated, by 14Krpm it is deposited on the glass fiber filter in the 1.5ml tubule immediately, remove supernatant carefully, settled material washed twice in the water of 1ml tissue culture level.
B. fast the preparation. shift out 500 μ l rapidly, and with Savant BIO 101Fast Prep Machine (Biocan Scientific) with speed 4 homogenate 20 seconds.As mentioned above with homogenate sedimentation and washing.
C. supernatant. after the vibration, allow sample leave standstill and made particulate matter be deposited to the pipe end in 5 minutes.Get 500 μ l supernatants sedimentation and washing as described above.
(BioRad Hercules CA) extracts RNA according to manufacturer's guidance from all samples with InstaGene Matrix.With ultraviolet spectrophotometry (A
260) measure the concentration of RNA, then 50ng is added in the PCR reaction mixture, its final concentration is the 2mM magnesium ion, 100 μ M dNTP, every kind of each 0.32 μ M of primer, and the 0.625 Taq Gold of unit polysaccharase.Universal primer p515f and p806r (Relman 1993) be used to the to increase fragment of about 300bp of 16S ribosomal RNA gene.Forward primer is modified by the sequence that is rich in GC with 40bp, stops the migration (.1989 such as Sheffield of amplified production on the different ureas/methane amide concentration of this sequence in denaturing gradient gel; .1993 such as Muyzer).Downcut target stripe from denaturant gel, and the amplified production of purifying is carried out cycle sequencing, cycle sequencing uses the condition (PE Applied Biosystems) of recommending to reinstate BigDye Terminator extension from reverse primer.On 310 Genetic Analyser (PE Applied Biosystems), carry out sequencing.With basic local comparison research tool (Basic Local Alignment Search Tool) (BLAST; Altschul etc., 1990).
The result
For same sample, any of three kinds of sample treatments all produces a kind of different characteristic pattern, as shown in Figure 1.Choose 9 significant bands and be used for order-checking.The partial sequence of the 16S rRNA gene of listed bacterial species has nearest coupling in 300bp fragment that is checked order and the BLAST result bar.In order to identify more accurately, may also need to measure bigger 16S rRNA fragment.
Table 3 has shown the blast search result's of the 300bp 16S rRNA gene fragment that is checked order summary.% homology between numeral unknown nucleotide sequence in the bracket and their the nearest coupling.
Table 3
| Band | The order-checking source | The band that mobility is identical | BLAST result |
| 1 | SM45-prepares fast | The SN45-vibration, C1 (1998)-vibration C/C (1998)-vibration | Sulfobacillus thermosulfidooxidans (98%) |
| 2 | C1 (1998)-vibration | SM45-prepares SN45-vibration C/C (1998)-vibration fast | Sulfobacillus thermosulfidooxidans (98%) |
| 8 | RH14K (60 ℃)-supernatant | PO45-prepares fast/vibrates/supernatant SS45-supernatant | Unknown bacterium (97%) denitrogenation Fe＜II〉oxidizing bacteria (97%) |
| 9 | 014A (50 ℃)-supernatant | SN45-prepares fast/vibrates/and supernatant SM45-prepares fast | Thiobacillus ferrooxidant (96%) |
| The 014A vibration |
Some bacterial species be saved or be replaced to imagination can so that it is worked under different temperature from top listed mixed culture.
For example, can use a kind of sulfur-oxidizing bacteria at a lower temperature---thiobacillus thiooxidans replaces Thiobacillus caldus.
The contriver thinks at least some are unusual in the PRELIMINARY RESULTS of the CHARACTERISTICS IDENTIFICATION gained of bacterial cultures of the present invention.Shown in following embodiment 2, bacterial cultures of the present invention has been carried out further CHARACTERISTICS IDENTIFICATION work.Because the improvement of search engine and the renewal of database, As time goes on the accuracy of such CHARACTERISTICS IDENTIFICATION work can improve.
Embodiment 2
Method
Sample to bacterial cultures of the present invention carries out denaturing gradient gel electrophoresis (" DGGE ").This method is at first described (1993) by Muyzer etc., and it is specially adapted to flora to complexity and carries out feature and describe.
With the bacterial cultures of 6 10mL equal portions～13, centrifugal 15 minutes of 000g.Use aforesaid method (Plumb etc., 2001) improved form, from centrifugation, extract gross sample DNA.Sample is precipitated in the phosphate buffered saline (PBS) that is resuspended in pH 7.2 with the trigger cell cracking.Come lysing cell further by (SDS) handling cell with lyase, N,O-Diacetylmuramidase and Proteinase K and strong stain remover sodium laurylsulfonate (sodium dodecylsulphate).Behind twice of phenol-chloroform-primary isoamyl alcohol extracting sample, the DNA in the solution is precipitated with Virahol and sodium acetate.The DNA that extracts further uses UltraCleanTM PCR Clean-up Kit (MO BIO Laboratories Inc.) purifying.After electrophoresis on the 1%w/v sepharose, make the DNA sample as seen with ethidium bromide staining.
From each DNA sample, with the 16S rRNA gene of polymerase chain reaction (PCR) amplification total length.To the special PCR primer of bacterium and archeobacteria (Archaea) and HotStarTaq polysaccharase (Qiagen) is common uses, as described in the forefathers (Plumb etc., 2002).PCR product UltraCleanTM PCR Clean-up Kit purifying, the primer that reacts as DGGE PCR then.The described primer sets of DGGE PCR use forefathers (Muyzer etc., 1993, vre s etc., 1997) carry out.Also use from three kinds with reference to bacterial strain: the DNA of JP2 strain that ferricoxidans, sulfobacillus thermosulfidooxidans and sulfolobus solfataricus belong to certain kind of (Sulfolobus) produces and is used for PCR fragment that DGGE analyzes so that comparison to be provided.DGGE uses the general sudden change detection system of DcodeTM, and (BioRadLaboratories, USA), 6%w/v polyacrylamide gel, denatured gradient were 30% to 70% (100% denaturing agent contains 7M urea and 40%v/v methane amide).Electrophoresis carries out 16h under 60 ℃, 100V.Gel dyes in the 1 * TAE damping fluid that contains 0.5mg L-1 ethidium bromide, and uses MultiImage
TMLight box formula transilluminator TM-26 (Alpha innotech Corporation, the U.S.) and Chemilmage software records.The band of selecting is downcut from gel, and carry out pcr amplification once more with the DGGE primer.The PCR product of purifying carries out sequencing with automated cycle order-checking, as described in the forefathers (Plumb etc., 2002).Sequencing result is compared research tool (Basic LocalAlignment Search Tool) (BLAST with basic part; Altschul etc., 1990) analyze, with the sequence data in sequence and the nonredundancy nucleic acid sequence data storehouse relatively, this database can be by http://www.ncbi.nlm.nih.gov/BLAST/ visit.
The result
Find the little bar-shaped cell of minority with the phase microscope observation sample.From 6 10mL samples each has all successfully extracted DNA.At the DNA purification step 6 samples are compiled, the result obtains the DNA sample of 3 purifying.
From the genomic dna of purifying, use bacterium specificity and the specific primer amplification of archeobacteria to go out the 16S rDNA of total length.This result's demonstration exists bacterium and archeobacteria simultaneously.With these PCR product purifications, and it goes out to be used for the dna fragmentation that DGGE analyzes as template by pcr amplification.Use bacterium specificity and the specific primer of archeobacteria to carry out PCR, successfully amplified the dna fragmentation that is used for the DGGE analysis.
Analyze the PCR fragment with DGGE then, they are separated according to the electrophoretic mobility of dna fragmentation on the gel matrix that contains cumulative denaturing agent concentration.From the PCR sample demonstration of reference strain as the band characteristic pattern of expecting.During the inoculum sample fragment that produced with the specific primer analysis of bacterium, two dimnesses have been obtained but distinctive band.During the sample fragment that produced with the specific primer analysis of bacterium, on gel, only produced a distinctive band.The zone that other part right and wrong are distinctive on the characteristic pattern, outward appearance is fuzzy.
To analyzing to determine the identity of these dna sequence dnas from the PCR product order-checking of target stripe and with BLAST.Table 4 has been summed up the result of the BLAST analysis of sequence data.Strips A contains and the DNA that comes the DNA highly similar (99%) of self-vulcanizing Bacillaceae.Band B and C contain and DNA from the DNA highly similar (98-99%) of the unknown strains of Thermoplasma.Thermoplasma comprises some biology of archeobacteria circle, it is characterized in that: owing to the polymorphism of the cellular form that does not have cell walls to cause, and can be in the ability of growing in the thermophilic temperature range by temperature in having a liking for.The representative of this genus is the acidophilic bacteria that can carry out heterotrophic growth under the condition of aerobic and anaerobic.
Table 4
The BLAST of the sequence data of the DNA band that downcuts from the DGGE collection of illustrative plates of sample analyzes.
| The band numbering | Sample message (primer) | Nearest coupling (% homology) |
| A | Inoculum (bacterium) | Sulfuration Bacillaceae bacterial classification G2 (99%) |
| B | Inoculum (bacterium) | Thermoplasma bacterial classification clone ASL1 (99%) |
| C | Inoculum (archeobacteria) | Thermoplasma bacterial classification clone ASL1 (98%) |
Unexpectedly, have the representative that has sulfuration Bacillaceae and Thermoplasma in the bacterial cultures of oxidation of sulfureted ore and refined ore ability simultaneously subject to the foregoing, also might add one or more bacteriums that embodiment 1 is identified.
According to imagination, can comprise base metal ore and refined ore (copper, nickel, cobalt zinc etc.) with the material that mixt bacteria culture of the present invention is handled, precious metal ore and refined ore (Jin Heyin) and platinum metals (PGM) ore and refined ore.Further imagine, this culture can be used for dump leaching, slot type leaching, the bucket formula leaches or hillock leaches oxidation.
Bacterial cultures of the present invention and method can be worked in wide temperature range, so just can save the relevant cost of cooling bacterial oxidation system.The chalcopyrite of all right oxidation form of ownership of this method, and used grinding particle size can not cause bigger fund and running cost.
Modifications and variations, for example conspicuous to those skilled in the art modifications and variations are considered to fall into scope of the present invention.
Reference
Altschul, S.F., W.Gish, W.Miller, E.W.Myers and D.J.Lipman.1990.Basiclocal alignment search tool.J.Mol.Bio.215:403-410.
Brierley C.L. and R Brans, 1994-Selection of BacTech ' s ThermophilicBio-Oxidation Process for Youanmi Mine, In Biomine 94, ConferenceProceeding Perth Western Australia.Section 5.
Barrett, J., Hughes, M.N., Ewart, D.K. and Poole, R.K., 1988-The isolation andcharacterisation of a moderately thermophilic mixed culture of autotrophicbacteria:application of the oxidation of refractory gold concentrates.Perth Gold88, Randol International Ltd, Golden, Colorado, pp 148 150.
Dew，D.M.and D.M.Miler，1997-The BioNIC Process；Bioleaching of MineralSulphide Concentrates For Recovery of Nickel，In IBS Biomine’97，ConferenceProceedings，Sydney，p M7.1.0-M7.1.9.
Muyzer, G.E.C.DeWall, and A.G.Uitterlinden, 1993.Profiling of complexmicrobial populations by denaturing gradient gel electrophoresis analysis ofpolymerase chain reaction amlified genes coding for the 16s rRNA.Appl.Environ.Microbiol.59:695-700.
Nobar, A.M., Ewart, D.K., Alsaffar, L., Barrett, J., Hughes M.N. and Poole, R.K., 1988-Isolation and characterisation of a mixed microbial community from anAustralian mine:application to the leaching of gold from refractory ores, Biohydrometallurgy (P.R.Norris and D.P.Kelly compile), Science and TechnologyLetters, Kew Surrey, UK, pp 530-531.
vre s, L., Forney, L., Daae, F.l. and Torsvik, V.1997.Distribution ofbacterioplankton in meromictic Lake S lenvannet, as determined by denaturinggradient gel electrophoresis of PCR-amplified gene fragments coding for 16SrRNA.Applied and Environmental Microbiology, 63,3367-3373.
Plumb, J.J., Bell, J. and Stuckey, D.C.2001.Microbial populations associated withtreatment of an industrial dye effluent in an anaerobic baffled reactor.Appliedand Environmental Microbiology, 67,3226-3235.
Plumb, J.J, Gibbs, B., Stoll, M.B., Robertson, W.J., Gibson, J.A.E., Nichols, P.D., Watling, H.R. and Franzmann, P.D.2002.Enrichment and characterisation ofthermophilic aidophiles for the bioleaching of mineral sulphides.MineralsEngineering, 15,787-794.
Relman, D.A.1993.Universal bacterial 16s rRNA amplification and sequencing, p489-495. at D.H.Persing, T.F.Smith, F.C.Tenover, and J.White (volume) DiagnosticMolecular Biology Principles and Applications-1993.American Society forMicrobiology, Washington is among the DC.
Sheffield, V.C., D.R.Cox, L.S.Lerman and R.M.Muyers.1989.Attachment of a40base pair G+C rich sequence (GC-clamp) to genomic RNA fragments bypolymerase chain reaction results in improved detection of single base changes.Proc.Natl.Acad.Sci USA 86:232-236.
Claims (9)
1. the bacterial cultures that is used for the bacterial oxidation of sulfide ore or refined ore, this bacterial cultures be identified by AGAL preservation registration number NM99/07541 or by adaptation and come, this bacterial cultures also contains one or more bacterial strains of sulfuration Bacillaceae and Thermoplasma simultaneously.
2. the bio-oxidation process of sulfide ore and refined ore, it is characterized in that using the bacterial cultures that identified by AGAL preservation registration number NM99/07541 or by and adapt to and the bacterial cultures that comes leaches this ore or refined ore, this bacterial cultures also contains one or more bacterial strains of sulfuration Bacillaceae and Thermoplasma simultaneously.
3. according to the technology of claim 2, it is characterized in that described sulfide ore or refined ore contain chalcopyrite.
4. according to the technology of claim 2 or 3, it is characterized in that leaching the form of carrying out and be selected from the group of forming by following:
Dump leaching,
Drill traverse goes out,
The bucket formula leach and
Hillock leaches.
5. according to each technology of claim 2 to 4, it is characterized in that this bacterial cultures be non-indigenous for wanting oxidized ore or refined ore.
6. according to each technology of claim 2 to 5, it is characterized in that the ore or the refined ore that are provided have grinding or the grinding particle size that is equal to or greater than P8575 μ m.
7. according to each technology of claim 2 to 5, it is characterized in that the ore or the refined ore that are provided have grinding or the grinding particle size that is equal to or greater than P8090 μ m.
8. according to each bacterial cultures of aforementioned claim, it is characterized in that this culture can work in 45 ℃ to 90 ℃ temperature range in the oxidation of sulfide ore and refined ore.
9. according to each bacterial cultures of claim 1 to 7, it is characterized in that this culture can work in 45 ℃ to 65 ℃ temperature range in the oxidation of sulfide ore and refined ore.
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| US10/734,581 US7189527B2 (en) | 1999-09-03 | 2003-12-15 | Bacterial oxidation of sulphide ores and concentrates |
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| CN101705286B (en) * | 2009-12-01 | 2012-03-07 | 北京有色金属研究总院 | Primer pair for amplifying sulfobacillus 16S rRNA gene and real-time PCR quantitative detection method in the environment thereof |
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| PE20071046A1 (en) | 2005-03-21 | 2007-12-21 | Bioheap Ltd | LEACHING BY SULFIDE MINERALS STACKS |
| CN100362117C (en) * | 2006-03-23 | 2008-01-16 | 福州大学 | Sulfide ore treatment technology by thermoacidophile |
| AP2268A (en) | 2006-05-12 | 2011-08-03 | Bhp Billiton Sa Ltd | Chloride heap leaching. |
| US8932809B2 (en) * | 2010-01-19 | 2015-01-13 | Opgen, Inc. | Methods and kits for isolating nucleic acid from an organism |
| CN101956071B (en) * | 2010-10-31 | 2012-04-11 | 中南大学 | Biological metallurgy mineral leaching microorganism combined bacterium fluid for copper ore and method for recycling metallic copper |
| CN103572049B (en) * | 2013-11-19 | 2015-07-01 | 东北大学 | Bacterium agitation leaching method of cobalt concentrate |
| US20160273060A1 (en) * | 2013-11-20 | 2016-09-22 | Biosigma S.A. | Bacterial strain, cutipay, isolated from the sulfobacillus thermosulfidooxidans species, bacterial inoculum comprising same, and method for the bioleaching of minerals where said bacterial inoculum is inoculated |
| EP3034635B1 (en) * | 2014-12-15 | 2018-10-31 | Middle East Mine and Industry Company | Tank bioleaching of copper sulfide ores |
| RU2692341C1 (en) * | 2018-12-28 | 2019-06-24 | Николай Игоревич Агафонов | Method for complex extraction of group 1 and group 8 metals |
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| US4729788A (en) | 1987-01-23 | 1988-03-08 | Advanced Mineral Technologies, Inc. | Thermophilic microbial treatment of precious metal ores |
| US4888293A (en) * | 1987-07-10 | 1989-12-19 | Giant Bay Biotech Inc. | Adapting bacteria to low pH and high arsenic concentration for use in oxidizing sulfide ores |
| US5089412A (en) * | 1987-07-10 | 1992-02-18 | Gb Biotech Inc. | Bacteria for oxidizing multimetallic sulphide ores |
| ZA922051B (en) | 1991-03-22 | 1993-09-20 | Bac Tech Australia | Bacterial oxidation of metal containing materials |
| PE11095A1 (en) | 1993-05-25 | 1995-05-08 | Mim Holdings Ltd | INTEGRATED BIOLOGICAL LEACHING PROCESS / SOLVENT EXTRACTION PROCESS FOR THE PRODUCTION OF ZINC METAL FROM ZINC CONCENTRATES |
| US5827701A (en) * | 1996-05-21 | 1998-10-27 | Lueking; Donald R. | Method for the generation and use of ferric ions |
| AR012179A1 (en) | 1997-03-27 | 2000-09-27 | Billiton Sa Ltd | A PROCEDURE FOR THE RECOVERY OF COPPER |
| US5873927A (en) | 1997-05-16 | 1999-02-23 | Echo Bay Mines, Limited | Integrated, tank/heap biooxidation process |
| AU749366B2 (en) | 1997-07-31 | 2002-06-27 | M.I.M. Holdings Limited | Silver catalysed leaching of chalcopyrite ore |
| AUPP444298A0 (en) * | 1998-07-01 | 1998-07-23 | Bactech (Australia) Pty Limited | Leaching of low sulphur ores |
| US6110253A (en) * | 1998-12-14 | 2000-08-29 | Geobiotics, Inc. | High temperature heap bioleaching process |
| AUPQ265199A0 (en) * | 1999-09-03 | 1999-09-30 | Pacific Ore Technology Limited | Improved bacterial oxidation of sulphide ores and concentrates |
| PE20020912A1 (en) * | 2000-11-25 | 2002-10-19 | Billiton Sa Ltd | BIOPRODUCT PRODUCTION |
| AUPR355101A0 (en) * | 2001-03-06 | 2001-04-05 | Pacific Ore Technology (Australia) Ltd | A method for the bacterially assisted heap leaching of chalcopyrite |
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| CN100381592C (en) | 2008-04-16 |
| AR047567A1 (en) | 2006-01-25 |
| CL2004003148A1 (en) | 2010-01-29 |
| AU2004297289A1 (en) | 2005-06-23 |
| US20040206208A1 (en) | 2004-10-21 |
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| EP1702080A4 (en) | 2008-07-23 |
| ZA200604848B (en) | 2007-11-28 |
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| EA200601156A1 (en) | 2006-12-29 |
| WO2005056842A1 (en) | 2005-06-23 |
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| CA2549656C (en) | 2013-09-17 |
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| CA2549656A1 (en) | 2005-06-23 |
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